Prime editing enables precise genome editing in mouse liver and retina

Jang, Hyewon; Shin, Jeong Hong; Jo, Dong Hyun; Seo, Jung Hwa; Yu, Goosang; Gopalappa, Ramu; Cho, Sung Rae; Kim, Jeong Hun; Kim, Hyongbum

doi:10.1101/2021.01.08.425835

Cited by 8 publications

(12 citation statements)

References 29 publications

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“…In our study, systemic injection of a single dose of AdV5 encoding the prime editing components into neonates resulted in an average of 58% editing at the Dnmt1 and 8% editing at the Pah enu2 locus. These rates are substantially higher than in recently reported prime editing studies in somatic tissues (20,45), and led to the restoration of physiological blood-L-Phe levels in Pah enu2 mice. Importantly, correction rates of approximately 10% in hepatocytes should also be sufficient to treat a variety of other genetic liver diseases, including tyrosinemia and urea cycle disorders (46)(47)(48).…”

Section: Discussionmentioning

confidence: 60%

Treatment of a metabolic liver disease by in vivo prime editing in mice

Boeck

Rothgangl

Villiger

et al. 2021

Preprint

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Prime editing is a highly versatile CRISPR-based genome editing technology with the potential to correct the vast majority of pathogenic mutations. However, correction of a disease phenotype in vivo in somatic tissues has not been demonstrated thus far. Here, we establish proof-of-concept for in vivo prime editing and repair the metabolic liver disease phenylketonuria (PKU) in mice. We first developed a size-reduced SpCas9 prime editor (PE) lacking the RNaseH domain of the reverse transcriptase (PE2-deltaRnH), and a linker- and NLS-optimized intein-split PE construct (PE2 p.1153) for delivery by adeno-associated virus (AAV) vectors. Systemic dual AAV-mediated delivery of this variant into the liver of neonatal mice enabled installation of a transversion mutation at the Dnmt1 locus with an average efficiency of 15%, and delivery of unsplit PE2-deltaRnH using human adenoviral vector 5 (AdV5) further increased editing rates to 58%. PE2-deltaRnH-encoding AdV5 was also used to correct the disease-causing mutation of the phenylalanine hydroxylase (Pah)enu2 allele in phenylketonuria (PKU) mice with an average efficiency of 8% (up to 17.3%), leading to therapeutic reduction of blood phenylalanine (L-Phe) levels. Our study demonstrates in vivo prime editing in the liver with high precision and editing rates sufficient to treat a number of metabolic liver diseases, emphasizing the potential of prime editing for future therapeutic applications.

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Section: Discussionmentioning

confidence: 60%

Treatment of a metabolic liver disease by in vivo prime editing in mice

Boeck

Rothgangl

Villiger

et al. 2021

Preprint

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“…Although the editing efficiency was modest at the Fah disease locus, we reached therapeutic thresholds for HT1. Furthermore, the initial editing efficiency of the AAV9 delivered Nme2-ABE8e-U6 construct exceeded that reported previously using other precision genome editing tools and delivery approaches [65][66][67]70]. Future optimization of this system promises to improve efficiency.…”

Section: Discussionmentioning

confidence: 78%

Adenine Base Editing in vivo with a Single Adeno-Associated Virus Vector

Zhang

Bamidele

et al. 2021

Preprint

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Base editors (BEs) have opened new avenues for the treatment of genetic diseases. However, advances in delivery approaches are needed to enable disease targeting of a broad range of tissues and cell types. Adeno-associated virus (AAV) vectors remain one of the most promising delivery vehicles for gene therapies. Currently, most BE/guide combinations and their promoters exceed the packaging limit (~5 kb) of AAVs. Dual-AAV delivery strategies often require high viral doses that impose safety concerns. In this study, we engineered an adenine base editor using a compact Cas9 from Neisseria meningitidis (Nme2Cas9). Compared to the well-characterized Streptococcus pyogenes Cas9-containing ABEs, Nme2-ABE possesses a distinct PAM (N4CC) and editing window, exhibits fewer off-target effects, and can efficiently install therapeutically relevant mutations in both human and mouse genomes. Importantly, we showed that in vivo delivery of Nme2-ABE and its guide RNA by a single-AAV vector can revert the disease mutation and phenotype in an adult mouse model of tyrosinemia. We anticipate that Nme2-ABE, by virtue of its compact size and broad targeting range, will enable a range of therapeutic applications with improved safety and efficacy due in part to packaging in a single-vector system.

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“…In terms of off-targeting ratio, researchers frequently identify susceptible off-targeting sites and perform the analysis using techniques such as NGS ( Jang et al, 2021 ), circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq) ( Levy et al, 2020 ; Suh et al, 2021 ), and nickase-based Digenome sequencing (nDigenome seq) ( Kim et al, 2020 ). With that, the off-targeting rates can be often underestimated since only few predicted off-targeting sites are studied.…”

Section: Crispr-based Gene Editing: a Brief Overviewmentioning

confidence: 99%

“…Similarly, a preprint by Jang et al showed an editing efficiency of 1.87% in the Atp7b locus in transduced mouse retina (no cell sorting) and no detectable indels were found. Jang and others used a trans -splicing AAV8 vector, which allows the expression of a single transcript encoded by two independent vectors coadministered to the same tissue to deliver the PE to the retina via intravitreal injection, in conjunction with an additional AAV8 construct to deliver the pegRNA and sgRNA ( Jang et al, 2021 ).…”

Section: Crispr-based Gene Editing: a Brief Overviewmentioning

confidence: 99%

Prime Editing for Inherited Retinal Diseases

et al. 2021

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Inherited retinal diseases (IRDs) are chronic, hereditary disorders that lead to progressive degeneration of the retina. Disease etiology originates from a genetic mutation—inherited or de novo—with a majority of IRDs resulting from point mutations. Given the plethora of IRDs, to date, mutations that cause these dystrophies have been found in approximately 280 genes. However, there is currently only one FDA-approved gene augmentation therapy, Luxturna (voretigene neparvovec-rzyl), available to patients with RPE65-mediated retinitis pigmentosa (RP). Although clinical trials for other genes are underway, these techniques typically involve gene augmentation rather than genome surgery. While gene augmentation therapy delivers a healthy copy of DNA to the cells of the retina, genome surgery uses clustered regularly interspaced short palindromic repeats (CRISPR)-based technology to correct a specific genetic mutation within the endogenous genome sequence. A new technique known as prime editing (PE) applies a CRISPR-based technology that possesses the potential to correct all twelve possible transition and transversion mutations as well as small insertions and deletions. EDIT-101, a CRISPR-based therapy that is currently in clinical trials, uses double-strand breaks and nonhomologous end joining to remove the IVS26 mutation in the CEP290 gene. Preferably, PE does not cause double-strand breaks nor does it require any donor DNA repair template, highlighting its unparalleled efficiency. Instead, PE uses reverse transcriptase and Cas9 nickase to repair mutations in the genome. While this technique is still developing, with several challenges yet to be addressed, it offers promising implications for the future of IRD treatment.

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Prime editing enables precise genome editing in mouse liver and retina

Cited by 8 publications

References 29 publications

Treatment of a metabolic liver disease by in vivo prime editing in mice

Treatment of a metabolic liver disease by in vivo prime editing in mice

Adenine Base Editing in vivo with a Single Adeno-Associated Virus Vector

Prime Editing for Inherited Retinal Diseases

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